The Role And Mechanism Of MAD2B In Cisplatin-induced Acute Kidney Injury

Objective Acute kidney injury is a common syndrome in clinic.Many causes can lead to acute kidney injury,including renal ischemia,infection,and nephrotoxic substances.Cisplatin is a commonly used tumor chemotherapy drug in clinic.However,it often causes acute kidney injury due to its nephrotoxicity.Therefore,it is of great significance to study the pathological mechanism of cisplatin-induced acute kidney injury.MAD2 B has been proven to play a role in various kidney diseases by regulating cell cycle progression,cell phenotypic transition and cell activation.However,its role in cisplatin-induced acute kidney injury remains unclear.The purpose of this study was to explore the role and mechanism of MAD2 B in cisplatin-induced acute kidney injury and to provide a new therapeutic target for the serious disease.Methods1.Cisplatin-induced acute kidney injury mouse model was established by single intraperitoneal injection of cisplatin.2.HK-2 cells cultured in vitro were treated with cisplatin to simulate cisplatin-induced acute kidney injury in vitro.3.HK-2 cells were pretreated with MAD2 B knockdown lentivirus or MAD2 B overexpression plasmid,and then treated with cisplatin to explore the effect of MAD2 B in cisplatin-induced HK-2 cells injury.4.Proximal tubule epithelial cell-specific MAD2 B knockout mice were constructed using Cre-Lox P system.To explore the effect of MAD2 B in cisplatin-induced acute kidney injury,MAD2 B knockout mice and wild-type littermates were simultaneously injected with cisplatin to induce acute kidney injury.5.HK-2 cells treated with MAD2 B knockdown lentivirus were pretreated with APC/C inhibitor and then treated with cisplatin to explore the effect of APC/C inhibitor on MAD2B sh RNA-transfected cells.6.Mice were intragastric treated with APC/C inhibitor and then intraperitoneal injection of cisplatin to induce acute kidney injury to explore the role of APC/C inhibitor in cisplatin-induced acute kidney injury.7.Levels of serum creatinine,urea nitrogen and albumin were measured by automatic chemical analyzer.8.The kidney injury was evaluated by HE staining.9.The protein levels of MAD2 B,MDM2,p53,and so on in mouse kidneys and HK-2 cells were detected by immunohistochemical staining,immunofluorescence staining or western blot.10.The RNA expressions of MDM2 and p53 in HK-2 cells were detected by quantitative polymerase chain reaction.11.The interaction between APC2 and MDM2 in cisplatin-treated HK-2 cells was detected by immunoprecipitation assay.12.The cell cycle status of HK-2 cells were detected by flow cytometry.13.Statistical analysis was performed using Graph Pad Prism 8 software.Results1.The expression of MAD2 B was upregulated in cisplatin-induced acute kidney injury mice and in cisplatin-treated HK-2 cells.Knockdown of MAD2 B aggravated HK-2 cells injury induced by cisplatin,while overexpression of MAD2 B alleviated the cells injury.Meanwhile,proximal tubule epithelial cell-specific MAD2 B knockout aggravated cisplatin-induced acute kidney injury in mice.2.Knockdown of MAD2 B promoted the increase of p53 induced by cisplatin and aggravated G1 phase arrest and apoptosis of the HK-2 cells,while overexpression of MAD2 B inhibited the increase of p53 and alleviated G1 phase arrest and apoptosis.Meanwhile,proximal tubule epithelial cell-specific MAD2 B knockout promoted the level of p53 and aggravates G1 phase arrest and apoptosis of tubule epithelial cells.3.MAD2 B knockdown inhibited the expression of MDM2 in HK-2 cells,while MAD2 B overexpression promoted the MDM2 level.Meanwhile,proximal tubular epithelial cell-specific MAD2 B knockout inhibited the expression of MDM2 in tubules.The APC/C inhibitor promoted the expression of MDM2,further alleviating p53-mediated cell cycle arrest and apoptosis and alleviating cisplatin-induced acute kidney injury.Moreover,it inhibited the regulatory effects of MAD2 B knockdown on expressions of MDM2,p53 and downstream molecules.ConclusionsMAD2B is elevated in cisplatin-induced acute kidney injury and alleviates acute kidney injury by inhibiting APC/C-targeted degradation of MDM2,thereby alleviating p53-mediated cell cycle arrest and apoptosis.